Clinically-observed FOXA1 mutations upregulate SEMA3C through transcriptional derepression in prostate cancer.

FOXA1 is a pioneer transcription factor that is frequently mutated in prostate, breast, bladder, and salivary gland malignancies. Indeed, metastatic castration-resistant prostate cancer (mCRPC) commonly harbour FOXA1 mutations with a prevalence of 35%. However, despite the frequent recurrence of FOXA1 mutations in prostate cancer, the mechanisms by which FOXA1 variants drive its oncogenic effects are still unclear. Semaphorin 3C (SEMA3C) is a secreted autocrine growth factor that drives growth and treatment resistance of prostate and other cancers and is known to be regulated by both AR and FOXA1. In the present study, we characterize FOXA1 alterations with respect to its regulation of SEMA3C. Our findings reveal that FOXA1 alterations lead to elevated levels of SEMA3C both in prostate cancer specimens and in vitro. We further show that FOXA1 negatively regulates SEMA3C via intronic cis elements, and that mutations in FOXA1 forkhead domain attenuate its inhibitory function in reporter assays, presumably by disrupting DNA binding of FOXA1. Our findings underscore the key role of FOXA1 in prostate cancer progression and treatment resistance by regulating SEMA3C expression and suggest that SEMA3C may be a driver of growth and tumor vulnerability of mCRPC harboring FOXA1 alterations.

Semaphorin 3C promotes de novo steroidogenesis in prostate cancer cells.

Intratumoral androgen biosynthesis contributes to castration-resistant prostate cancer progression in patients treated with androgen deprivation therapy. The molecular mechanisms by which castration-resistant prostate cancer acquires the capacity for androgen biosynthesis to bypass androgen deprivation therapy are not entirely known. Here, we show that semaphorin 3C, a secreted signaling protein that is highly expressed in castration-resistant prostate cancer, can promote steroidogenesis by altering the expression profile of key steroidogenic enzymes. Semaphorin 3C not only upregulates enzymes required for androgen synthesis from dehydroepiandrosterone or de novo from cholesterol but also simultaneously downregulates enzymes involved in the androgen inactivation pathway. These changes in gene expression correlate with increased production of androgens induced by semaphorin 3C in prostate cancer model cells. Moreover, semaphorin 3C upregulates androgen synthesis in LNCaP cell-derived xenograft tumors, likely contributing to the enhanced in vivo tumor growth rate post castration. Furthermore, semaphorin 3C activates sterol regulatory element-binding protein, a transcription factor that upregulates enzymes involved in the synthesis of cholesterol, a sole precursor for de novo steroidogenesis. The ability of semaphorin 3C to promote intratumoral androgen synthesis may be a key mechanism contributing to the reactivation of the androgen receptor pathway in castration-resistant prostate cancer, conferring continued growth under androgen deprivation therapy. These findings identify semaphorin 3C as a potential therapeutic target for suppressing intratumoral steroidogenesis.

Dependency of Tamoxifen Sensitive and Resistant ER+ Breast Cancer Cells on Semaphorin 3C (SEMA3C) for Growth.

Estrogen receptor positive (ER+) breast cancer (BCa) accounts for the highest proportion of breast cancer-related deaths. While endocrine therapy is highly effective for this subpopulation, endocrine resistance remains a major challenge and the identification of novel targets is urgently needed. Previously, we have shown that Semaphorin 3C (SEMA3C) is an autocrine growth factor that drives the growth and treatment resistance of various cancers, but its role in breast cancer progression and endocrine resistance is poorly understood. Here, we report that SEMA3C plays a role in maintaining the growth of ER+ BCa cells and is a novel, tractable therapeutic target for the treatment of ER+ BCa patients. Analyses of publicly available clinical datasets indicate that ER+ BCa patients express significantly higher levels of SEMA3C mRNA than other subtypes. Furthermore, SEMA3C mRNA expression was positively correlated with ESR1 mRNA expression. ER+ BCa cell lines (MCF7 and T47D) expressed higher levels of SEMA3C mRNA and protein than a normal mammary epithelial MCF10A cell line. ER siRNA knockdown was suppressed, while dose-dependent beta-estradiol treatment induced SEMA3C expression in both MCF7 and T47D cells, suggesting that SEMA3C is an ER-regulated gene. The stimulation of ER+ BCa cells with recombinant SEMA3C activated MAPK and AKT signaling in a dose-dependent manner. Conversely, SEMA3C silencing inhibited Estrogen Receptor (ER) expression, MAPK and AKT signaling pathways while simultaneously inducing apoptosis, as monitored by flow cytometry and Western blot analyses. SEMA3C silencing significantly inhibited the growth of ER+ BCa cells, implicating a growth dependency of ER+ BCa cells on SEMA3C. Moreover, the analysis of tamoxifen resistant (TamR) cell models (TamC3 and TamR3) showed that SEMA3C levels remain high despite treatment with tamoxifen. Tamoxifen-resistant cells remained dependent on SEMA3C for growth and survival. Treatment with B1SP Fc fusion protein, a SEMA3C pathway inhibitor, attenuated SEMA3C-induced signaling and growth across a panel of tamoxifen sensitive and resistant ER+ breast cancer cells. Furthermore, SEMA3C silencing and B1SP treatment were associated with decreased EGFR signaling in TamR cells. Here, our study implicates SEMA3C in a functional role in ER+ breast cancer signaling and growth that suggests ER+ BCa patients may benefit from SEMA3C-targeted therapy.

Targeting Semaphorin 3C in Prostate Cancer With Small Molecules.

Despite the amenability of early-stage prostate cancer to surgery and radiation therapy, locally advanced and metastatic prostate cancer is clinically problematic. Chemical castration is often used as a first-line therapy for advanced disease, but progression to the castration-resistant prostate cancer phase occurs with dependable frequency, largely through mutations to the androgen receptor (AR), aberrant AR signaling, and AR-independent mechanisms, among other causes. Semaphorin 3C (SEMA3C) is a secreted signaling protein that is essential for cardiac and neuronal development and has been shown to be regulated by the AR, to drive epithelial-to-mesenchymal transition and stem features in prostate cells, to activate receptor tyrosine kinases, and to promote cancer progression. Given that SEMA3C is linked to several key aspects of prostate cancer progression, we set out to explore SEMA3C inhibition by small molecules as a prospective cancer therapy. A homology-based SEMA3C protein structure was created, and its interaction with the neuropilin (NRP)-1 receptor was modeled to guide the development of the corresponding disrupting compounds. Experimental screening of 146 in silico‒identified molecules from the National Cancer Institute library led to the discovery of four promising candidates that effectively bind to SEMA3C, inhibit its association with NRP1, and attenuate prostate cancer growth. These findings provide proof of concept for the feasibility of inhibiting SEMA3C with small molecules as a therapeutic approach for prostate cancer.

Polyclonal HIV envelope-specific breast milk antibodies limit founder SHIV acquisition and cell-associated virus loads in infant rhesus monkeys.

Breast milk HIV-1 transmission is currently the predominant contributor to pediatric HIV infections. Yet, only ~10% of breastfeeding infants born to untreated HIV-infected mothers become infected. This study assessed the protective capacity of natural HIV envelope-specific antibodies isolated from the milk of HIV-infected women in an infant rhesus monkey (RM), tier 2 SHIV oral challenge model. To mimic placental and milk maternal antibody transfer, infant RMs were i.v. infused and orally treated at the time of challenge with a single weakly neutralizing milk monoclonal antibody (mAb), a tri-mAb cocktail with weakly neutralizing and ADCC functionalities, or an anti-influenza control mAb. Of these groups, the fewest tri-mAb-treated infants had SHIV detectable in plasma or tissues (2/6, 5/6, and 7/8 animals infected in tri-mAb, single-mAb, and control-mAb groups, respectively). Tri-mAb-treated infants demonstrated significantly fewer plasma transmitted/founder variants and reduced peripheral CD4+ T cell proviral loads at 8 weeks post-challenge compared to control mAb-treated infants. Abortive infection was observed as detectable CD4+ T cell provirus in non-viremic control mAb- and single mAb-, but not in tri-mAb-treated animals. These results suggest that polyfunctional milk antibodies contribute to the natural inefficiency of HIV-1 transmission through breastfeeding and infant vaccinations eliciting non-neutralizing antibody responses could reduce postnatal HIV transmission.

SEMA3C drives cancer growth by transactivating multiple receptor tyrosine kinases via Plexin B1.

Growth factor receptor tyrosine kinase (RTK) pathway activation is a key mechanism for mediating cancer growth, survival, and treatment resistance. Cognate ligands play crucial roles in autocrine or paracrine stimulation of these RTK pathways. Here, we show SEMA3C drives activation of multiple RTKs including EGFR, ErbB2, and MET in a cognate ligand-independent manner via Plexin B1. SEMA3C expression levels increase in castration-resistant prostate cancer (CRPC), where it functions to promote cancer cell growth and resistance to androgen receptor pathway inhibition. SEMA3C inhibition delays CRPC and enzalutamide-resistant progression. Plexin B1 sema domain-containing:Fc fusion proteins suppress RTK signaling and cell growth and inhibit CRPC progression of LNCaP xenografts post-castration in vivo SEMA3C inhibition represents a novel therapeutic strategy for treatment of advanced prostate cancer.

Semaphorin 3 C drives epithelial-to-mesenchymal transition, invasiveness, and stem-like characteristics in prostate cells.

Prostate cancer (PCa) is among the most commonly-occurring cancers worldwide and a leader in cancer-related deaths. Local non-invasive PCa is highly treatable but limited treatment options exist for those with locally-advanced and metastatic forms of the disease underscoring the need to identify mechanisms mediating PCa progression. The semaphorins are a large grouping of membrane-associated or secreted signalling proteins whose normal roles reside in embryogenesis and neuronal development. In this context, semaphorins help establish chemotactic gradients and direct cell movement. Various semaphorin family members have been found to be up- and down-regulated in a number of cancers. One family member, Semaphorin 3 C (SEMA3C), has been implicated in prostate, breast, ovarian, gastric, lung, and pancreatic cancer as well as glioblastoma. Given SEMA3C's roles in development and its augmented expression in PCa, we hypothesized that SEMA3C promotes epithelial-to-mesenchymal transition (EMT) and stem-like phenotypes in prostate cells. In the present study we show that ectopic expression of SEMA3C in RWPE-1 promotes the upregulation of EMT and stem markers, heightened sphere-formation, and cell plasticity. In addition, we show that SEMA3C promotes migration and invasion in vitro and cell dissemination in vivo.

Androgen receptor transcriptionally regulates semaphorin 3C in a GATA2-dependent manner.

The androgen receptor (AR) is a member of the nuclear receptor superfamily of transcription factors and is central to prostate cancer (PCa) progression. Ligand-activated AR engages androgen response elements (AREs) at androgen-responsive genes to drive the expression of gene batteries involved in cell proliferation and cell fate. Understanding the transcriptional targets of the AR has become critical in apprehending the mechanisms driving treatment-resistant stages of PCa. Although AR transcription regulation has been extensively studied, the signaling networks downstream of AR are incompletely described. Semaphorin 3C (SEMA3C) is a secreted signaling protein with roles in nervous system and cardiac development but can also drive cellular growth and invasive characteristics in multiple cancers including PCa. Despite numerous findings that implicate SEMA3C in cancer progression, regulatory mechanisms governing its expression remain largely unknown. Here we identify and characterize an androgen response element within the SEMA3C locus. Using the AR-positive LNCaP PCa cell line, we show that SEMA3C expression is driven by AR through this element and that AR-mediated expression of SEMA3C is dependent on the transcription factor GATA2. SEMA3C has been shown to promote cellular growth in certain cell types so implicit to our findings is the discovery of direct regulation of a growth factor by AR. We also show that FOXA1 is a negative regulator of SEMA3C. These findings identify SEMA3C as a novel target of AR, GATA2, and FOXA1 and expand our understanding of semaphorin signaling and cancer biology.

PTEN loss promotes mitochondrially dependent type II Fas-induced apoptosis via PEA-15.

Two distinct biochemical signals are delivered by the CD95/Fas death receptor. The molecular basis for the differential mitochondrially independent (type I) and mitochondrially dependent (type II) Fas apoptosis pathways is unknown. By analyzing 24 Fas-sensitive tumor lines, we now demonstrate that expression/activity of the PTEN tumor suppressor strongly correlates with the distinct Fas signals. PTEN loss-of-function and gain-of-function studies demonstrate the ability to interconvert between type I and type II Fas pathways. Importantly, from analyses of Bcl-2 transgenic Pten(+/-) mice, Pten haploinsufficiency converts Fas-induced apoptosis from a Bcl-2-independent to a Bcl-2-sensitive response in primary thymocytes and activated T lymphocytes. We further show that PTEN influences Fas signaling, at least in part, by regulating PEA-15 phosphorylation and activity that, in turn, regulate the ability of Bcl-2 to suppress Fas-induced apoptosis. Thus, PTEN is a key molecular rheostat that determines whether a cell dies by a mitochondrially independent type I versus a mitochondrially dependent type II apoptotic pathway upon Fas stimulation.

Recombinant Mycobacterium bovis bacillus Calmette-Guerin elicits human immunodeficiency virus type 1 envelope-specific T lymphocytes at mucosal sites.

A successful vaccine vector for human immunodeficiency virus type 1 (HIV-1) should induce anti-HIV-1 T-cell immune responses at mucosal sites. We have constructed recombinant Mycobacterium bovis bacillus Calmette-Guérin (rBCG) expressing an HIV-1 group M consensus envelope (Env) either as a surface, intracellular, or secreted protein as an immunogen. rBCG containing HIV-1 env plasmids engineered for secretion induced optimal Env-specific T-cell gamma interferon enzyme-linked immunospot responses in murine spleen, female reproductive tract, and lungs. While rBCG-induced T-cell responses to HIV-1 envelope in spleen were lower than those induced by adenovirus prime/recombinant vaccinia virus (rAd-rVV) boost, rBCG induced comparable responses to rAd-rVV immunization in the female reproductive tract and lungs. T-cell responses induced by rBCG were primarily CD4(+), although rBCG alone did not induce anti-HIV-1 antibody. However, rBCG could prime for a protein boost by HIV-1 envelope protein. Thus, rBCG can serve as a vector for induction of anti-HIV-1 consensus Env cellular responses at mucosal sites.

Generation of mucosal anti-human immunodeficiency virus type 1 T-cell responses by recombinant Mycobacterium smegmatis.

A successful vaccine vector for human immunodeficiency virus type 1 (HIV-1) should induce anti-HIV-1 immune responses at mucosal sites. We have generated recombinant Mycobacterium smegmatis vectors that express the HIV-1 group M consensus envelope protein (Env) as a surface, intracellular, or secreted protein and have tested them in animals for induction of both anti-HIV-1 T-cell and antibody responses. Recombinant M. smegmatis engineered for expression of secreted protein induced optimal T-cell gamma interferon enzyme-linked immunospot assay responses to HIV-1 envelope in the spleen, female reproductive tract, and lungs. Unlike with the induction of T-cell responses, priming and boosting with recombinant M. smegmatis did not induce anti-HIV-1 envelope antibody responses, due primarily to insufficient protein expression of the insert. However, immunization with recombinant M. smegmatis expressing HIV-1 Env was able to prime for an HIV-1 Env protein boost for the induction of anti-HIV-1 antibody responses.

Capric acid and hydroxypropylmethylcellulose increase the immunogenicity of nasally administered peptide vaccines.

Immunization by the nasal route is an established method for the induction of mucosal and systemic humoral and cell-mediated antigen-specific responses. However, the effectiveness of nasal immunization is often hampered by the need for increased doses of antigen. Bioadhesives and absorption enhancers were investigated for their ability to enhance immune responses in mice after nasal immunization with model HIV-1 peptide and protein immunogens. Two additives, hydroxypropylmethylcellulose (HPMC) and capric acid, consistently enhanced antigen-specific serum IgG endpoint titers under conditions in which antigen dose was limiting. Nasal immunization of mice with 20 microg of an HIV-1 peptide immunogen plus cholera toxin (CT) as adjuvant induced serum antipeptide IgG titers of 1:9.5log2 after four immunizations while the addition of CA or HPMC to the vaccine formulation increased serum antipeptide IgG titers to 1:15.4log2 and 1:17.6log2, respectively. When 5 microg recombinant HIV-1 gp41 was used as the immunogen, the addition of CA or HPMC to the vaccine formulation increased serum anti-gp41 IgG titers to 1:11.6log2 and 1:8.8log2, respectively, compared to 1:5.2log2 after three nasal immunizations with 5 microg gp41 + CT alone. Thus, HPMC and capric acid may be useful additives that increase the immunogenicity of nasally administered vaccines and permit less antigen to be used with each immunization.

Inhibition of the phosphatidylinositol 3'-kinase pathway promotes autocrine Fas-induced death of phosphatase and tensin homologue-deficient prostate cancer cells.

Rationally designed therapeutics that target the phosphatidylinositol 3'-kinase (PI3K) cell survival pathway are currently in preclinical and clinical development for cancer therapy. Drugs targeting the PI3K pathway aim to inhibit proliferation, promote apoptosis, and enhance chemosensitivity and radiosensitivity of cancer cells. The phosphatase and tensin homologue (PTEN) phosphatidylinositol 3'-phosphatase is a key negative regulator of the PI3K pathway. Inactivation of the PTEN tumor suppressor results in constitutive activation of the PI3K pathway and is found in approximately 50% of advanced prostate cancers, which correlates with a high Gleason score and poor prognosis. Inhibition of the PI3K pathway leads to apoptosis of prostate cancer cells; however, the precise mechanism by which this occurs is unknown. Here we report that apoptotic cell death of PTEN-deficient LNCaP and PC3 prostate cancer cells induced by the PI3K inhibitor LY294002 can be abrogated by disrupting Fas/Fas ligand (FasL) interactions with recombinant Fas:Fc fusion protein or FasL neutralizing antibody (Nok-1), or by expressing dominant-negative Fas-associated death domain. Furthermore, we find that apoptosis induced by expression of wild-type PTEN, driven by a tetracycline-inducible expression system in LNCaP cells, can be inhibited by blocking Fas/FasL interaction using Fas:Fc or Nok-1. These data show that apoptosis induced by blockade of the PI3K pathway in prostate tumor cells is mediated by an autocrine Fas/FasL apoptotic mechanism and the Fas apoptotic pathway is both necessary and sufficient to mediate apoptosis by PI3K inhibition.

Loss of PTEN is associated with progression to androgen independence.

BACKGROUND: Progression to a lethal androgen-independent (AI) stage of advanced prostate cancer is a critical clinical obstacle limiting patient survival. PTEN inactivation is frequently observed in advanced prostate cancer and correlates with a poor prognosis. However, the functional significance of PTEN inactivation in AI progression has not been demonstrated. METHODS: PTEN expression was examined in benign, hormone naïve and AI human prostate cancer specimens, and in recurrent AI Shionogi tumors. The effect of antisense oligonucleotide (ASO)-mediated PTEN downregulation in AI progression of the Shionogi tumor model was determined. RESULTS: Significantly reduced PTEN expression was observed in AI versus benign and hormone naïve prostate tumors. Seven of 14 AI Shionogi tumors exhibited marked downregulation or complete loss of PTEN. ASO-mediated PTEN inhibition reduced androgen-withdrawal induced regression of Shionogi tumors and accelerated AI progression. CONCLUSIONS: These data suggest that PTEN inactivation may play a role in progression to androgen independence.

Microarray analysis reveals vegetative molecular phenotypes of Arabidopsis flowering-time mutants.

The transition to flowering occurs at the shoot apex; however, most of the characterized genes that affect the timing of floral induction are expressed throughout the plant. To further our understanding of these genes and the flowering process, the vegetative molecular phenotypes of 16 Arabidopsis mutants associated with the major flowering initiation pathways were assayed using a 13,000 clone microarray under two different conditions that affect flowering. All mutants showed at least one change in gene expression other than the mutant flowering gene. Metabolism- and defence-related pathways were the areas with the most frequent gene expression changes detected in the mutants. Several genes such as EARLI1 were differentially expressed in a number of flowering mutants from different flowering pathways. Analysis of the promoter regions of genes differentially expressed identified common promoter elements, indicating some form of common regulation.

Gender differences in human immunodeficiency virus type 1-specific CD8 responses in the reproductive tract and colon following nasal peptide priming and modified vaccinia virus Ankara boosting.

Induction of mucosal anti-human immunodeficiency virus type 1 (HIV-1) T-cell responses in males and females will be important for the development of a successful HIV-1 vaccine. An HIV-1 envelope peptide, DNA plasmid, and recombinant modified vaccinia virus Ankara (rMVA) expressing the H-2D(d)-restricted cytotoxic T lymphocyte P18 epitope were used as immunogens to test for their ability to prime and boost anti-HIV-1 T-cell responses at mucosal and systemic sites in BALB/c mice. We found of all prime-boost combinations tested, an HIV-1 Env peptide subunit mucosal prime followed by systemic (intradermal) boosting with rMVA yielded the maximal induction of gamma interferon (IFN-gamma) spot-forming cells in the female genital tract and colon. However, this mucosal prime-systemic rMVA boost regimen was minimally immunogenic for the induction of genital, colon, or lung anti-HIV-1 T-cell responses in male mice. We determined that a mucosal Env subunit immunization could optimally prime an rMVA boost in female but not male mice, as determined by the magnitude of antigen-specific IFN-gamma responses in the reproductive tracts, colon, and lung. Defective mucosal priming in male mice could not be overcome by multiple mucosal immunizations. However, rMVA priming followed by an rMVA boost was the optimal prime-boost strategy for male mice as determined by the magnitude of antigen-specific IFN-gamma responses in the reproductive tract and lung. Thus, prime-boost immunization strategies able to induce mucosal antigen-specific IFN-gamma responses were identified for male and female mice. Understanding the cellular and molecular basis of gender-determined immune responses will be important for optimizing induction of anti-HIV-1 mucosal immune responses in both males and females.

Exacerbation of experimental autoimmune encephalomyelitis in rodents infected with murine gammaherpesvirus-68.

Viral infections have long been suspected to play a role in the pathogenesis of multiple sclerosis. In the present study, two different rodent models of experimental autoimmune encephalomyelitis (EAE) were used to demonstrate the ability of murine gammaherpesvirus-68 (gammaHV-68) to exacerbate development of neurological symptoms. SJL mice received UV-inactivated gammaHV-68 or intranasalgammaHV-68, followed by immunization against proteolipid-protein peptide 139-151. Infected mice became moribund within 10 days post-immunization, whereas mice exposed to UV-inactivated gammaHV-68 recovered. In the second model, Lewis rats were exposed to UV-inactivated gammaHV-68 or to gammaHV-68, followed by passive transfer of encephalitogenic T lymphocytes specific for myelin basic protein. Consistently, infected rats had higher clinical scores, and this result was observed during acute or latent gammaHV-68 infection. It is unlikely that this gammaHV-68-induced exacerbation was due to significant viral replication within the central nervous system since nested PCR, viral plaque assays, and infectious-centers assays demonstrated no detectable virus in spinal cords or brains of infected rodents undergoing EAE. Taken together, these studies demonstrate increased clinical symptoms of EAE in rodents infected by a gammaherpesvirus that has a limited ability to invade the central nervous system.

An iAc/Ds gene and enhancer trapping system for insertional mutagenesis in rice.

We evaluated a two-component transposon iAc/Ds system for generating a library of insertional mutants in rice. The constructs used have gene or enhancer trapping properties, plasmid rescue and T-DNA/Ds launching pad reporter facilities. Mutagenic iAc/Ds lines were produced by three methods: crossing iAc and Ds containing lines; co-transformation with iAc and Ds constructs; and super-transformation of iAc transgenic calli with Ds constructs. First and second generation screening populations, derived from crosses (F2 and F3) or double transformation (DtT1 and DtT2), were analysed for stable insertion lines containing Ds transposed to locations unlinked to iAc. The average frequencies of putative stable insertion (PSI) lines in the F2, DtT1, F3 and DtT2 populations were 6.61, 5.58, 11.47 and 7.05% respectively, with large variations in these frequencies in screening populations derived from different mutagenic lines. Further analyses indicated that 41, 33, 65 and 64% of the PSI lines, respectively, have Ds transposed to locations unlinked to the original Ds launching pad. Using the plasmid rescue system, sequences flanking Ds from 137 PSI lines were obtained. Sixty-eight of these lines had unique insertions in genomic regions, of which 18 were known sequences. Because the average frequency of proven stable insertion lines in any of our screening populations has been less than 5%, we suggest that additional features should be incorporated in this two-component iAc/Ds system to increase the screening efficiency, and to make it suitable for large-scale insertional mutagenesis and determination of gene function in rice.